Heart disease is the leading cause of human mortality and morbidity. The ubiquitin-proteasome system (UPS) is pivotal to protein quantity and quality control in the cell. UPS dysregulation, especially proteasome functional insufficiency, plays a major role in the progression from a large subset of heart diseases to heart failure and, accordingly, proteasome enhancement is implicated as a new strategy to treat heart disease with increased proteotoxic stress (IPTS). To develop pharmacological means to enhance the proteasome, however, requires understanding how proteasome activity is regulated as such regulatory mechanisms could potentially be exploited to enhance the proteasome. Recent advances in cell biology show that phosphorylation of the proteasome often increases proteasome activities but the in vivo physiological significance of proteasome phosphoregulation has not been established. Thus, the goal of this project is to advance our understanding on how specific proteasome phosphorylation regulates cardiac physiology and pathophysiology. Our pilot studies have confirmed genetically in mice that phosphorylation of RPN6/PSMD11 at Ser14 is responsible for proteasome activation by cAMP-dependent protein kinase (PKA). Our preliminary data further revealed that (1) myocardial Ser14-phopshorylated Rpn6 (referred to as p-Rpn6) was markedly altered in mice with inherited IPTS and mice subjected to myocardial ischemia or trans-aortic constriction (TAC) and (2) genetic blockade and mimicry of p-Rpn6 substantially mitigated cardiac responses to various stressors. Hence, we propose to test the central hypotheses that p-Rpn6 is essential to 26S Psm activation to meet the increased demand for selective proteolysis in stressed cardiac muscle, via pursuit of these specific aims: (1) to determine the necessity of p-Rpn6 in cardiac proteostasis and cardiac function at baseline, (2) to determine the role of increased p-Rpn6 in the inherited heart disease with IPTS, and (3) to determine the role of increased p-Rpn6 in an acquired heart disease with IPTS. New mouse models created with gene editing to block or mimic p-Rpn6, as well as p-Rpn6 specific antibodies will be used along with a well-established UPS performance reporter. Tandem mass-tags (TMT) based multiplexing coupled with tandem mass spectrometry will be used to profile ubiquitinomes shaped by p-Rpn6 in stressed hearts. This research will provide the ultimate in vivo demonstration for the molecular basis of PKA-elicited proteasome activation, determine unequivocally for the first time the (patho)physiological significance of this key proteasome phosphoregulation in intact animals, and illustrate whether this regulation can be exploited for therapeutic purposes.